This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Signaling via small cytosolic GTPase [unreadable]RhoA lies at the crossroads of many important signaling cascades, regulating processes such as smooth muscle contraction, neuronal morphogenesis, and reorganization of actin cytoskeleton. Activity of RhoA is tightly regulated by a sophisticated interplay between two proteins [unreadable]guanine nucleotide exchange factor (RhoGEF), GTPase-activating protein (RhoGAP). GEFs catalyze the exchange of a guanine nucleotide on RhoA, and consequently cause RhoA activation. The aim of this research is to characterize the mechanism of regulation of two RhoA-specific GEFs - leukemia-associated RhoGEF (LARG) and PDZ-RhoGEF (PRG). It is assumed that GEFs in their inactive state are autoinhibited by the mutual disposition of their four domains [unreadable]PDZ, RGSL, DH, and PH, and that the activation of the protein involves rearrangements of proteins` supramodular architecture. The high resolution structures of individual GEF domains are known, however there is no structural information about the full-length protein. Studies of these large molecules are difficult, because many portions of interdomain linkers are expected to be unstructured, and therefore such proteins are recalcitrant to crystallization. On the other hand, their size makes it virtually impossible to use NMR techniques. We therefore used small-angle X-ray scattering (SAXS), which is increasingly recognized as a powerful tool in the characterization of multidomain proteins in solution, to probe the structure of LARG and PRG in their autoinhibited and constitutively active conformations, and in complex with their downstream effector - RhoA.